ES2779851T3 - Mixture of fatty acids (F.A.G., group of fatty acids) for use in the treatment of inflammatory pathologies - Google Patents

Abstract

Pharmaceutical, cosmetic and / or dietary composition comprising a mixture of at least five fatty acids selected from palmitic acid, oleic acid, stearic acid, linoleic acid, alpha-linolenic acid, gamma-linolenic acid, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA ), azelaic acid and myristic acid and the physiologically stable excipient N-2-hydroxyethylpalmitamide.

Description

DESCRIPTION

Mixture of fatty acids (F.A.G., group of fatty acids) for use in the treatment of inflammatory pathologies

State of the art

Fatty acids are aliphatic carboxylic acids and represent the constituent components of almost all complex lipids and vegetable and animal fats. These compounds can be classified according to the length of the carbon chain and / or the presence of double bonds in said chain (saturated, unsaturated fatty acids).

In particular, some unsaturated fatty acids are considered essential because the body does not synthesize them, but must be supplemented through diet. Among these, some acids are distinguished according to the position of the last double bond on the carbon chain, such as omega-3, where the last double bond is on the third carbon from the end (for example, linolenic acid), omega-6 where the last double bond is on the sixth carbon from the end (for example, linoleic acid) or, in addition, omega-9 where the last double bond is on the ninth carbon from the end (for example , oleic acid).

In 1964, Bergstrom, Samuelsson et al. (Nutrition classics. Biochimica et Biophysica Acta 90: 207-10, 1964. The enzymatic formation of prostaglandin E2 from arachidonic acid. Prostaglandins and related factors 32) demonstrated the role and biological effects of lipids in the inflammatory process and in other diseases .

In 1979, the first phospholipid was discovered, specifically phosphatidylinositol, along with its role in activating cellular response processes, said phospholipid being capable of activating and controlling reactivity through messaging elements.

Lipids, therefore, play different roles in the body, including acting as chemical messengers that can cause changes in the role of an individual cell or determine actions that can change the microenvironment, as in the case of the processes involved in the response. to inflammatory attacks. Lipids in esterified form can fit on the membrane and be transported as signal elements to other cells. These compounds can also bind to certain proteins and remain inactive until they reach the site of action and bind to the appropriate receptor. Essential fatty acids, such as linoleic acid and linolenic acid, are known to be precursors of arachidonic acid present in membrane phospholipids (for example, those of the cell membrane) and of many different types of eicosanoids, that is, substances involved in the body's inflammatory response, including hydroxyeicosatetraenoic acids, prostanoids (prostaglandins, thromboxanes and prostacyclins), leukotrienes, lipoxins and resolvins, chemical mediators that play an important role in pain, fever, edema, blood clotting and, more generally, inflammation.

As is known, the inflammatory response is a multifactorial physiological reaction characterized by the participation of different cells of the immune system, for example mast cells, macrophages, basophils and / or lymphocytes, with different intervention times.

One of the first cells that intervenes in the inflammatory process is the mast cell, whose ability to respond and trigger the inflammatory process is of the order of microseconds. Its activation generates a series of reactions that result in the release of preformed mediators contained within its cytoplasm; in rapid succession, macrophages are called and activated.

The function of macrophages is structured in two phases: the first, known as M1, involves the activation of a series of reactions that result in the release into the microenvironment of chemical mediators, such as NGF (nerve growth factor), VEGF (vascular endothelial growth factor), FGF (fibroblast growth factor), histamine, interleukins, cytokines, and lipid products, such as arachidonic acid, prostaglandins, and heparin, which can trigger and support the inflammatory process, as well as "attract" the other cells of the immune system to the site of inflammation. The second phase, known as M2, involves the activation of the "sequestrant" phenomenon aimed at eliminating the waste resulting from the destruction of the agent responsible for the inflammatory action. Between the macrophage activation phase in M1 and M2, basophils, whose function is to release histamine in the microenvironment subjected to the aggression of the inflammatory agent, are attracted and activated, which leads to vasodilation and, consequently, edema as a result of diapédesis or extravasation of immune cells.

When the inflammatory reaction has reached the diapédesis phase, the lymphocytes, whose role is to counteract the pathogen, reach the area. The entire inflammatory process is triggered in just a few seconds.

All complex biological systems are regulated by an opposition system based on agonism and antagonism mechanisms. More generally, degranulation of mast cell mediators triggers a series of phenomena that are synthesized during the inflammatory process. The cells of the immune system mentioned above are self-regulating by fine receptor mechanisms involving a sophisticated system of receptors, expressed in the cytoplasmic membrane, which can be overexpressed during stimulation processes.

This overexpression determines a release, within the extracellular space, of a series of chemical mediators, which trigger a series of events whose purpose is to defend the tissue microenvironment and provoke repair phenomena. The system is regulated by the production of receptor antagonists, which are produced by the cell itself, from fatty acids taken from the cell membrane.

These biological systems are based on the control of receptors: after stimulation of the pathogen, cells express specific receptors that are saturated with mediators of their own production, that is, formed from the fatty acids that make up the membranes of said cells. Receptor expression is the means by which cells involved in the inflammatory process can transfer growth factors, interleukins, cytokines, etc., to the microenvironment. The saturation of these receptors allows first the reduction and then the modulation of degranulation of the mediators present within the cytoplasm of the cells involved in the inflammatory process (mast cells in particular) until the stimulation induced by the presence of the pathogen stops. .

However, this regulation means that it comes to an end when the continuous decrease in fatty acids in cell membranes causes suffering to the cell itself. In this condition, the receptors are overexpressed and, for the cell, this constitutes a degranulation signal destined for the mediators that triggers defense phenomena that are no longer needed.

Therefore, if there were no receptor control receptor, cells would obviously induce degranulation of everything present in the cytoplasm, leading to the attraction of other cells to the microenvironment. This would then lead to irritation of the system that, remaining active, could become a source of damage, leading to chronic inflammatory pathologies and autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis and systemic lupus erythematosus.

Therefore, it is very important that, in these pathological conditions, the body can control the hyperactive inflammatory process through the formation of a receptor antagonist consisting of fatty acids taken from the membrane of the cells themselves.

Currently, to meet this need, research has focused on dietary strategies designed to reduce the synthesis of pro-inflammatory chemical mediators, such as prostaglandins, by reducing the consumption of vegetable oils and fatty meats, and by increasing the intake of fish and certain oils. in particular, such as flax and hemp. With this method, it is thought that higher amounts of eicosapentaenoic acid and docosahexaenoic acid (omega-3) would be incorporated into the membrane phospholipids instead of arachidonic acid.

However, it is well known that these dietary strategies are not sufficient to eradicate serious inflammatory pathologies such as rheumatoid arthritis, chronic ulcerative colitis, systemic lupus erythematosus, pelvic inflammatory disease or atherosclerosis, and it is necessary to resort to pharmacological therapies.

Currently, pharmacological therapies used for inflammatory conditions include corticosteroids (such as cortisone and similar substances) or NSAIDs (non-steroidal anti-inflammatory drugs), which act at different levels of the arachidonic acid cascade. Corticosteroids counteract the release of arachidonic acid from phospholipids through inhibition of phospholipase activity, including phospholipase A2 (PLA2) and phospholipase C (PLC). In particular, the mechanism by which corticosteroids exert their anti-inflammatory and immunosuppressive action is highly structured and involves various biochemical processes implemented by the cell in response to potentially harmful stimuli (for example, infectious agents, allergens, foreign substances, abnormal cells, etc. .).

The role of this mechanism is to trigger an immune response, maintain it until the risk is eliminated, and then dampen the response so that it does not become harmful (as happens, for example, in cases of chronic inflammation or autoimmune diseases). In particular, corticosteroids inhibit cellular processes that lead to the synthesis of pro-inflammatory and immunostimulatory substances and, vice versa, activate cellular processes that lead to the synthesis of anti-inflammatory and immunosuppressive substances, in order to reduce the symptoms of the disease.

Aside from the anti-inflammatory / immunosuppressive effect, the side effects of synthesized corticosteroids generally stem from the fact that they interfere with the homeostatic systems of the body and thus can lead to: hypertension, water retention, hyperglycemia, potassium loss, osteoporosis , muscular hypertrophy, capillary fragility, delayed wound healing, hyperlipidemia, accumulation of adipose tissue in the face, neck and abdomen, gastroduodenal ulcers, increased blood coagulability, hematological disorders, euphoria and insomnia.

In addition, in prolonged treatment, these pharmacological substances tend to inhibit the production of analogous natural hormones by the adrenal glands, thus causing a situation of adrenal insufficiency, which occurs with serious consequences, especially after discontinuation of therapy. In addition, prolonged use of corticosteroids is related to their immunosuppressive action, which increases susceptibility to infections.

Meanwhile, NSAIDs interfere with the arachidonic acid cascade at a different level, inhibiting cyclooxygenases COX1 and 2, which are involved in inflammatory processes. The most common side effects are those that affect the digestive system, particularly the stomach, and include pain, burning sensations, or nausea, and ulceration of the gastric mucosa with possible bleeding; as well as skin reactions in susceptible individuals (erythema, urticaria).

Furthermore, document US2010 / 113387A1 describes a composition comprising myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and excipients for the treatment of viral infections, bacterial infections and inflammation. Document US2012 / 252888A1 describes a liquid pharmaceutical composition comprising DHA, EPA, linoleic acid, gamma linolenic acid and excipients, for the treatment of neurodegenerative disorders, multiple sclerosis and autoimmune diseases.

Document US2005 / 208162A1 describes a fatty acid composition comprising palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, myristic acid and excipients for the treatment and prevention of different types of arthritis and inflammatory conditions of the joints, periodontal diseases and psoriasis.

Document JP2009149599 A describes medicinal fats and oils comprising palmitic acid, oleic acid, stearic acid, linoleic acid, myristic acid and excipients for the treatment of inflammation, i.e. atopic dermatitis and document WO2011 / 027373A1 refers to a composition for pharmaceutical or veterinary use, comprising palmitoylethanolamide for human or veterinary use, containing a therapeutically effective amount of palmitoylethanolamide in ultra-micronized form, wherein more than 90% by weight of palmitoylethanolamide has particle sizes less than 6 microns, together with pharmaceutically acceptable excipients.

Therefore, there is a need to identify one or more compounds for the treatment of inflammation that can effectively block the hyperactivity of the inflammatory process, reducing the side effects associated with conventional treatments.

Brief description of the figures

Figure 1: Summary table containing the chemical structures of the fatty acids of the present invention.

Figure 2: Mechanism of action of macrophages

Figure 3: Determination of IC ⁵⁰ for AS66

Figure 4: Standard curve and percentage of inhibition of IL-8 with AS66

Figure 5: Standard curve and percentage of inhibition of IL-6 with AS66

Figure 6: Determination of IC ⁵⁰ for LC88

Figure 7: Standard curve and percentage of inhibition of IL-6 with LC88

Description

Surprisingly, it has been found that a mixture of specific fatty acids can effectively treat the inflammatory process, with improved control of the regulatory system and without side effects, through the biological modulation of the cells involved in the inflammatory process (Biological Modulation of inflammatory cells BMIC, "Biological Modulation of Inflammatory Cells").

A mixture of at least three fatty acids is described, containing from 8 to 24 carbon atoms, said fatty acids can be saturated, unsaturated or mixtures thereof.

The mixture preferably contains from three to ten fatty acids, and more preferably said mixture contains four, five, eight or nine fatty acids.

Said fatty acids are preferably selected from palmitic acid, oleic acid, stearic acid, linoleic acid, alpha-linolenic acid, gamma-linolenic acid, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), azelaic acid and myristic acid, or a mixture thereof.

The term "palmitic acid", according to this invention, refers to hexadecanoic acid, that is, a saturated monocarboxylic acid containing 16 carbon atoms (Figure 1).

The term "oleic acid", according to this invention, refers to cis-9-octadecenoic acid, that is, an unsaturated monocarboxylic acid (omega-9) containing 18 carbon atoms (Figure 1).

The term "stearic acid", according to this invention, refers to octadecanoic acid, that is, a saturated monocarboxylic acid containing 18 carbon atoms (Figure 1).

The term "linoleic acid," according to this invention, refers to cis, cis-9,12-octadecenoic acid, that is, an unsaturated monocarboxylic acid (omega-6) containing 18 carbon atoms (Figure 1).

The term "alpha-linolenic acid", according to this invention, refers to cis, cis, cis-9,12,15-octadecatrienoic acid, that is, an unsaturated monocarboxylic acid (omega-3) containing 18 carbon atoms ( Figure 1).

The term "gamma-linolenic acid", according to this invention, refers to cis, cis, cis-6,9,12-octadecatrienoic acid, that is, an unsaturated monocarboxylic acid (omega-6) containing 18 carbon atoms ( Figure 1).

The term "eicosapentaenoic acid (EPA)", according to this invention, refers to (5Z, 8Z, 11Z, 14Z, 17Z) -eicosa-5,8,11,14,17-pentenoic acid, that is, a monocarboxylic acid unsaturated (omega-3) containing 20 carbon atoms (Figure 1).

The term "docosahexaenoic acid (DHA)", according to this invention, refers to (4Z, 7Z, 10Z, 13Z, 16Z, 19Z) -docosa-4,7,10,13,16,19-hexaenoic acid, that is , an unsaturated monocarboxylic acid (omega-3) containing 22 carbon atoms (Figure 1).

The term "azelaic acid", according to this invention, refers to nonanedioic acid, that is, a saturated dicarboxylic acid containing 9 carbon atoms (Figure 1).

The term "myristic acid", according to this invention, refers to tetradecanoic acid, that is, a saturated monocarboxylic acid containing 14 carbon atoms (Figure 1).

Preferably, the mixture described contains six fatty acids, namely: palmitic acid, oleic acid, linoleic acid, alpha-linolenic acid, gamma-linolenic acid and eicosapentaenoic acid (EPA). Preferably, the described mixture contains seven fatty acids, namely: palmitic acid, oleic acid, stearic acid, linoleic acid, alpha-linolenic acid, and eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA).

More preferably, the mixture described contains four fatty acids, namely: palmitic acid, oleic acid, stearic acid and linoleic acid.

More preferably, the described mixture contains five fatty acids, namely: palmitic acid, oleic acid, stearic acid, linoleic acid and alpha-linolenic acid.

More preferably, said mixture contains eight fatty acids, namely: palmitic acid, oleic acid, stearic acid, linoleic acid, alpha-linolenic acid, gamma-linolenic acid, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA).

More preferably, said mixture contains nine fatty acids, namely: palmitic acid, oleic acid, stearic acid, linoleic acid, alpha-linolenic acid, gamma-linolenic acid, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and azelaic acid.

According to a description, palmitic acid is contained in the mixture in an amount comprised between 0.2% and 48% by weight; oleic acid is contained in the mixture in an amount comprised between 0.2% and 38% by weight; stearic acid is contained in the mixture in an amount comprised between 0.2% and 42% by weight; linoleic acid is contained in the mixture in an amount comprised between 0.2% and 40% by weight; alpha-linolenic acid is contained in the mixture in an amount comprised between 0.2% and 38% by weight; gamma-linoleic acid is contained in the mixture in an amount comprised between 0.2% and 30% by weight; eicosapentaenoic acid is contained in the mixture in an amount comprised between 0.2% and 25% by weight; docosahexaenoic acid is contained in the mixture in an amount comprised between 0.2% and 25% by weight; azelaic acid is contained in the mixture in an amount comprised between 0.2% and 40% by weight; myristic acid is contained in the mixture in an amount comprised between 0.005% and 0.01% by weight;

The percentages mentioned above are amounts by weight expressed with respect to the total weight of the mixture.

When the above-mentioned mixture contains four fatty acids, palmitic acid is contained in the above-mentioned mixture in an amount comprised between 0.2% and 48% by weight, preferably between 30% and 45% by weight, and more preferably up to about 40% of the total weight of the blend.

In the aforementioned mixture, oleic acid is contained in an amount comprised between 0.2% and 38% by weight, preferably between 15% and 30% by weight, and more preferably up to about 25% by weight. total mix.

In the aforementioned mixture, stearic acid is contained in an amount between 0.2% and 42% by weight, preferably between 20% and 35% by weight, and more preferably up to about 32% by weight. total mix.

In the above-mentioned mixture, linoleic acid is contained in an amount comprised between 0.2% and 38% by weight, preferably between 1% and 4% by weight, and more preferably up to about 3% by weight. total mix.

When the above-mentioned mixture contains five fatty acids, palmitic acid is contained in the above-mentioned mixture in an amount comprised between 0.2% and 48% by weight, preferably between 20% and 40% by weight, and more preferably up to about 38% of the total weight of the blend.

In the above-mentioned mixture, oleic acid is contained in an amount comprised between 0.2% and 38% by weight, preferably between 20% and 30% by weight, and more preferably up to about 27% by weight. total mix.

In the aforementioned mixture, stearic acid is contained in an amount between 0.2% and 42% by weight, preferably between 7% and 15% by weight, and more preferably up to about 10% by weight. total mix.

In the above-mentioned mixture, linoleic acid is contained in an amount comprised between 0.2% and 40% by weight, preferably between 8% and 20% by weight, and more preferably up to about 15% by weight. total mix.

In the above-mentioned mixture, alpha-linolenic acid is contained in an amount comprised between 0.2% and 38% by weight, preferably between 2% and 15% by weight, and more preferably up to about 10% of the total weight of the mixture.

When the above-mentioned mixture contains eight fatty acids, palmitic acid is contained in the above-mentioned mixture in an amount comprised between 0.2% and 48% by weight, preferably between 20% and 38% by weight, and more preferably up to about 30% of the total weight of the blend.

In the aforementioned mixture, oleic acid is contained in an amount comprised between 0.2% and 38% by weight, preferably between 10% and 15% by weight, and more preferably up to about 12% by weight. total mix.

In the aforementioned mixture, stearic acid is contained in an amount comprised between 0.2% and 42% by weight, preferably between 8% and 15% by weight, and more preferably up to about 12% by weight. total mix.

In the aforementioned mixture, linoleic acid is contained in an amount comprised between 0.2% and 40% by weight, preferably between 10% and 20% by weight, and more preferably up to about 18% by weight. total mix.

In the above-mentioned mixture, alpha-linolenic acid is contained in an amount between 0.2% and 38% by weight, preferably between 10% and 15% by weight, and more preferably up to about 12%. of the total weight of the mixture.

In the aforementioned mixture, gamma-linoleic acid is contained in an amount comprised between 0.2% and 30% by weight, preferably between 1% and 3% by weight, and more preferably up to about 2%. of the total weight of the mixture.

In the aforementioned mixture, eicosapentaenoic acid is contained in an amount comprised between 0.2% and 25% by weight, preferably between 5% and 10% by weight, and more preferably up to about 8% by weight. total mix.

In the above-mentioned mixture, docosahexaenoic acid is contained in an amount comprised between 0.2% and 25% by weight, preferably between 4% and 10% by weight, and more preferably up to about 6% by weight. total mix.

When the above-mentioned mixture contains nine fatty acids, palmitic acid is contained in the above-mentioned mixture in an amount comprised between 0.2% and 48% by weight, preferably between 20% and 30% by weight, and more preferably up to about 25% of the total weight of the blend.

In the aforementioned mixture, oleic acid is contained in an amount comprised between 0.2% and 38% by weight, preferably between 10% and 16% by weight, and more preferably up to about 14% by weight. total mix.

In the aforementioned mixture, stearic acid is contained in an amount comprised between 0.2% and 42% by weight, preferably between 8% and 15% by weight, and more preferably up to about 12% by weight. total mix.

In the above-mentioned mixture, linoleic acid is contained in an amount comprised between 0.2% and 40% by weight, preferably between 15% and 25% by weight, and more preferably up to about 20% by weight. total mix.

In the above-mentioned mixture, alpha-linolenic acid is contained in an amount between 0.2% and 38% by weight, preferably between 5% and 10% by weight, and more preferably up to about 8%. of the total weight of the mixture.

In the aforementioned mixture, gamma-linoleic acid is contained in an amount between 0.2% and 30% by weight, preferably between 2% and 6% by weight, and more preferably up to about 4% of the total weight of the mixture.

In the aforementioned mixture, eicosapentaenoic acid is contained in an amount comprised between 0.2% and 25% by weight, preferably between 5% and 10% by weight, and more preferably up to about 7% by weight. total mix.

In the aforementioned mixture, docosahexaenoic acid is contained in an amount comprised between 0.2% and 25% by weight, preferably between 5% and 10% by weight, and more preferably up to about 8% by weight. total mix.

In the aforementioned mixture, azelaic acid is contained in an amount comprised between 0.2% and 40% by weight, preferably between 1% and 3% by weight, and more preferably up to about 2% by weight. total mix.

According to one description, when the mixture contains four fatty acids, such as palmitic acid, oleic acid, stearic acid and linoleic acid, they are present in a weight ratio of about 1.6: 1: 1.28.0.12, respectively.

According to a preferred description, when the mixture contains five fatty acids, such as palmitic acid, oleic acid, stearic acid, linoleic acid and alpha-linolenic acid, they are present in a weight ratio of approximately 1.9: 1.35: 0 , 5: 0.75: 0.5, respectively.

According to a preferred description, when the mixture according to this invention contains eight fatty acids, such as palmitic acid, oleic acid, stearic acid, linoleic acid, alpha-linolenic acid, gamma-linolenic acid, eicosapentaenoic acid and docosahexaenoic acid, they are present in a weight ratio of about 2.4: 0.96: 0.96: 1.44: 0.96: 0.16: 0.64: 0.48 respectively.

According to a further preferred description, when the mixture according to this invention contains nine fatty acids, such as palmitic acid, oleic acid, stearic acid, linoleic acid, alpha-linolenic acid, gamma-linolenic acid, eicosapentaenoic acid, docosahexaenoic acid and azelaic acid, they are preferably present in a weight ratio of about 2.25: 1.26: 1.08: 1.80: 0.72: 0.36: 0.63: 0.72: 0.18 respectively.

Surprisingly, it has been observed that by establishing a set of the aforementioned fatty acids, in particular four, five, eight or nine fatty acids according to this invention, it has been possible to establish the control of cells involved in inflammatory processes, such as mast cells, macrophages, basophils, and lymphocytes.

This fatty acid delivery system has two important advantages:

1) a more rapid and reactive response to hyperstimulation induced by the inflammatory agent: membrane lipids in the cell do not decrease, thus resulting in temporary dispersion;

2) Significant energy saving: the cell does not have to use energy to recover lipids from the membranes and then replace them.

In fact, it has been observed that, by providing the set of fatty acids according to this invention, an improved control of the inflammatory process is obtained and the physiological conditions and the normal conditions of the microenvironment involved are restored in shorter times, all with respect to the technique previous.

A further description is a pharmaceutical, cosmetic and / or dietetic composition comprising the above mixture and at least one physiologically acceptable excipient.

A physiologically acceptable excipient is according to this invention any excipient known to a person skilled in the art that is useful in the preparation of pharmaceutical and / or cosmetic compositions.

The other excipients are generally classified according to the function they perform in the final composition. Preferable suitable excipients according to this invention include, for example, diluents, absorbents, glidants, binders, lubricants, surfactants, disintegrants, preservatives, antioxidants, or mixtures thereof.

Alternatively, excipients can be classified according to the functional group present in their chemical structure, such as sugars, amides, ethers, alcohols, and the like.

A particularly preferred excipient is N-2-hydroxyethylpalmitamide.

According to an embodiment of this invention, the aforementioned composition comprises a mixture of at least five fatty acids selected from the following: palmitic acid, oleic acid, stearic acid, linoleic acid, alpha-linolenic acid, gamma-linolenic acid, eicosapentaenoic acid ( EPA), docosahexaenoic acid (DHA), azelaic acid and myristic acid, and N-2-hydroxyethylpalmitamide.

According to an embodiment of this invention, palmitic acid is contained in the composition in an amount comprised between 5% and 45% by weight; oleic acid is contained in the composition in an amount comprised between 2% and 35% by weight; stearic acid is contained in the composition in an amount comprised between 2% and 35% by weight; linoleic acid is contained in the composition in an amount comprised between 1% and 40% by weight; alpha-linolenic acid is contained in the composition in an amount comprised between 1% and 25% by weight; gamma-linolenic acid is contained in the composition in an amount comprised between 0.5% and 10% by weight; eicosapentaenoic acid is contained in the composition in an amount comprised between 1% and 20% by weight; docosahexaenoic acid is contained in the composition in an amount comprised between 1% and 25% by weight; azelaic acid is contained in the composition in an amount comprised between 0.5% and 10% by weight; myristic acid is contained in the composition in an amount comprised between 0.05% and 1% by weight; the percentages mentioned above are amounts by weight expressed with respect to the total weight of the composition.

According to a description, said composition contains a mixture comprising four fatty acids and at least one pharmaceutically acceptable excipient.

When the mixture comprises four fatty acids, palmitic acid is contained in the composition in an amount between 2% and 40% by weight, preferably between 5% and 25% by weight, and more preferably up to about 6%. % of the total weight of the composition.

In the aforementioned composition, oleic acid is contained in an amount between 1% and 35% by weight, preferably between 2% and 10% by weight, and more preferably up to about 4% of the total weight of the composition.

In the aforementioned composition, stearic acid is contained in an amount comprised between 0.5% and 15% by weight, preferably between 2% and 10% by weight, and more preferably up to about 5% by weight. total composition.

In the aforementioned composition, linoleic acid is contained in an amount comprised between 0.5% and 5% by weight, preferably between 1% and 3% by weight, and more preferably up to about 1% by weight. total composition.

According to a preferred embodiment, said composition contains a mixture of five fatty acids, such as palmitic acid, oleic acid, stearic acid, linoleic acid and alpha-linolenic acid.

When the mixture of the present invention comprises five fatty acids, palmitic acid is contained in the composition in an amount comprised between 1% and 20% by weight, preferably between 3% and 10% by weight, and more preferably up to about 6% of the total weight of the composition.

In the aforementioned composition, oleic acid is contained in an amount between 1% and 15% by weight, preferably between 2% and 8% by weight, and more preferably up to about 4.5% by weight. total composition.

In the aforementioned composition, stearic acid is contained in an amount comprised between 0.5% and 7%, preferably between 1% and 5% by weight, and more preferably up to approximately 2% of the total weight of the composition.

In the aforementioned composition, linoleic acid is contained in an amount comprised between 0.5% and 10% by weight, preferably between 1% and 5% by weight, and more preferably up to about 2.5% of the total weight of the composition.

In the aforementioned composition, alpha-linolenic acid is contained in an amount comprised between 0.5% and 10% by weight, preferably between 1% and 6% by weight, and more preferably up to about 2% of the total weight of the composition.

According to a preferred embodiment of this invention, said composition contains a mixture comprising eight fatty acids and at least one pharmaceutically acceptable excipient.

When the mixture of the present invention comprises eight fatty acids, palmitic acid is contained in the composition in an amount comprised between 1% and 30% by weight, preferably between 3% and 10% by weight, and more preferably up to about 6% of the total weight of the composition.

In the aforementioned composition, oleic acid is contained in an amount comprised between 1% and 25% by weight, preferably between 5% and 15% by weight, and more preferably up to about 10% of the total weight of the composition.

In the aforementioned composition, stearic acid is contained in an amount comprised between 1% and 25% by weight, preferably between 5% and 15% by weight, and more preferably up to about 10% of the total weight of the composition.

In the aforementioned composition, linoleic acid is contained in an amount comprised between 0.2% and 10% by weight, preferably between 0.7% and 5% by weight, and more preferably up to about 1%. of the total weight of the composition.

In the aforementioned composition, alpha-linolenic acid is contained in an amount comprised between 0.2% and 6% by weight, preferably between 0.5% and 4% by weight, and more preferably up to about 1% of the total weight of the composition.

In the aforementioned composition, gamma-linolenic acid is contained in an amount comprised between 0.04% and 3% by weight, preferably between 0.2% and 1% by weight, and more preferably up to about 0.5% of the total weight of the composition.

In the aforementioned composition, eicosapentaenoic acid is contained in an amount comprised between 1% and 15% by weight, preferably between 2% and 6% by weight, and more preferably up to approximately 3% of the total weight of the composition.

In the aforementioned composition, docosahexaenoic acid is contained in an amount comprised between 0.5% and 7% by weight, preferably between 1% and 4% by weight, and more preferably up to about 2% by weight. total composition.

In a further preferred embodiment of this invention, said composition contains a mixture comprising nine fatty acids and at least one pharmaceutically acceptable excipient.

When the mixture of the present invention comprises nine fatty acids, palmitic acid is contained in the composition in an amount comprised between 1% and 30% by weight, preferably between 3% and 10% by weight, and more preferably up to about 7% of the total weight of the composition.

In the aforementioned composition, oleic acid is contained in an amount comprised between 1% and 25% by weight, preferably between 5% and 15% by weight, and more preferably up to about 10% of the total weight of the composition.

In the aforementioned composition, stearic acid is contained in an amount comprised between 1% and 25% by weight, preferably between 5% and 15% by weight, and more preferably up to about 10% of the total weight of the composition.

In the aforementioned composition, linoleic acid is contained in an amount comprised between 0.2% and 6% by weight, preferably between 0.5% and 3% by weight, and more preferably up to about 1%. of the total weight of the composition.

In the aforementioned composition, alpha-linolenic acid is contained in an amount comprised between 0.2% and 6% by weight, preferably between 0.5% and 4% by weight, and more preferably up to about 1% of the total weight of the composition.

In the aforementioned composition, gamma-linolenic acid is contained in an amount comprised between 0.04% and 3% by weight, preferably between 0.2% and 1% by weight, and more preferably up to about 0.5% of the total weight of the composition.

In the aforementioned composition, eicosapentaenoic acid is contained in an amount comprised between 1% and 15% by weight, preferably between 2% and 6% by weight, and more preferably up to approximately 3% of the total weight of the composition.

In the aforementioned composition, docosahexaenoic acid is contained in an amount comprised between 0.5% and 7% by weight, preferably between 1% and 4% by weight, and more preferably up to about 2% by weight. total composition.

In the aforementioned composition, azelaic acid is contained in an amount comprised between 0.05% and 2% by weight, preferably between 0.2% and 1% by weight, and more preferably up to about 0, 5% of the total weight of the composition.

The composition of this invention can be formulated in a form suitable for oral, topical, rectal, vaginal, ophthalmic, or parenteral administration.

In a preferred embodiment of this invention, said oral form can be selected from tablets, capsules, granules, oil capsules, solutions, suspensions, vehicles or nebulizer solutions, and more preferably is selected from capsules, tablets or solutions.

The capsule can be a soft gelatin capsule, a hard capsule or a capsule containing granules.

According to a further preferred embodiment of this invention, said topical form can be selected from cream, ointment, gel, solution, suspension, spray, patch or lyophilized granular powder, more preferably it is selected from cream, gel, spray, ointment and lyophilized granular powder.

According to a further preferred embodiment of this invention, said form suitable for vaginal administration is an ovum, a catheter, a gel or a solution for endocavitary use. According to a further preferred embodiment of this invention, said form suitable for rectal administration is a suppository, an enema or a solution for endocavitary use.

According to a further preferred embodiment of this invention, said form suitable for ophthalmic administration is eye drops, a wash, a cream or an ointment.

The term "wash" is understood as an eye wash solution.

According to another preferred embodiment of this invention, said form suitable for parenteral administration may be either an aqueous buffer solution or an oily suspension, and preferably said parenteral form is an oily suspension.

According to a further preferred embodiment of this invention, the aforementioned composition is a dermocosmetic composition, a pharmaceutical substance, a dietary supplement or a drug.

The fatty acid mixture in the present invention is preferably contained in the above-mentioned formulations in an amount ranging from 10% to 60% by weight, and preferably from 15% to 45% of the total weight of the formulation.

According to one description, the composition of the present invention containing a mixture of four fatty acids is preferably formulated topically.

According to one embodiment of this invention, the composition of the present invention containing a mixture of five fatty acids is preferably formulated topically or orally.

According to one embodiment of this invention, the composition of the present invention containing a mixture of eight fatty acids is preferably formulated in oral form, and more preferably in tablet or suspension form.

Said composition containing a mixture of eight fatty acids is preferably formulated parenterally or rectally.

According to a further embodiment of this invention, the composition of the invention containing a mixture of nine fatty acids is formulated orally, more preferably in the form of a tablet or suspension, or in topical form, and more preferably in the form of a cream. , gel form or ointment form.

In the aforementioned oral form containing said mixture of nine fatty acids, azelaic acid is contained in an amount comprised between 0.04% and 0.5% by weight, preferably between 0.08% and 0, 4% by weight, and more preferably up to about 0.2%.

In the aforementioned topical form containing said mixture of nine fatty acids, azelaic acid is contained in an amount comprised between 0.05% and 2% by weight, preferably between 0.2% and 1% by weight. , and more preferably up to about 0.5%.

According to the invention, the composition of the present invention can be administered to humans, intended to include both adults and the "pediatric population" (where the term "pediatric population" is understood as the part of the population that goes from birth to eighteen years of age), and animals.

A further object of the present invention is a mixture containing at least five fatty acids and / or a composition containing said mixture, for use in the treatment of inflammatory pathologies.

The inflammatory pathologies mentioned are of the acute or chronic variety and include: dermatological pathologies, such as atopic dermatitis, dermatomyositis, scleroderma, psoriasis, polymyositis, pemphigus, epidermolysis bullosa pemphigoid; ophthalmic pathologies, such as Sjogren's syndrome, sympathetic ophthalmia, uveitis and uveo-retinitis; pathologies of the mucosa, such as inflammation of the gastrointestinal mucous membranes (Crohn's disease) and inflammation of the oral and genital mucosa; joint and connective pathologies, such as rheumatoid arthritis, psoriatic arthritis, lupus erythematosus arthritis, and discoid and systemic lupus erythematosus; chronic pathological inflammations, such as chronic solar dermatitis, asthma and intestinal and pulmonary fibrosis, and chronic arthritis, degenerative pathologies that affect the peripheral nervous system (PNS) and the central nervous system (CNS), such as multiple sclerosis, autoimmune neurodegenerative pathologies, non-autoimmune neurodegenerative pathologies, inflammatory processes associated with the CNS, such as Parkinson's disease, senile dementia, bacterial meningitis, HIV infection and traumatic lesions, and PNS pathologies, such as radiculopathy caused by inflammation; pathologies of the central and peripheral nervous system where inflammatory processes follow the first ischemic insult, such as neuropathies due to compression, as well as traumatic neuropathies, strokes, and cranial trauma; cardiological diseases derived from perfusion phenomena as a consequence of ischemic injuries; inflammatory pathologies associated with fibrosis, such as allergic conjunctivitis, giant papillary conjunctivitis, food allergies, abnormal scarring, such as hypertrophic scar, keloids and ocular scar pemphigoid; pathologies in which kidney function is impaired as a result of inflammation of the kidneys. With respect to the administration to animals of the composition of the present invention, the local and / or systemic anti-inflammatory action exerted by the set of fatty acids is useful in the treatment of neurogenic inflammation (for example, spinal cord compression, lesions nervous disorders in dogs), joint and connective pathologies such as laminitis in horses (where the use of cortisone drugs is not possible), arthritis, respiratory pathologies, ophthalmic inflammation, keratoconjunctivitis sicca and inflammatory allergic manifestations, including food allergies.

The composition of this invention is preferably administered daily, both to humans and animals, within a range of one to four doses per day.

Said dose preferably contains from 0.1 to 50 mg of composition / kg of the patient's body weight, and more preferably from 0.5 to 20 mg / kg of the patient's body weight. According to a preferred embodiment of the present invention, said composition is administered for at least four weeks.

Experimental part

Examples

The following mixtures were prepared.

Example 1: (for reference only)

Eicosapentaenoic acid (EPA) ranging from 0.5% to 15%

Docosahexaenoic acid (DHA) ranging from 0.5% to 15%

Palmitic acid ranging from 0.5% to 35%

Oleic acid ranging from 0.5% to 35%

Stearic acid ranging from 0.5% to 15%

Gamma-linolenic acid ranging from 0.5% to 15%

Alpha-linolenic acid ranging from 0.5% to 15%

Azelaic acid ranging from 0.5% to 35%

- Linoleic acid ranging from 0.5% to 35%

Example 2: (for reference only)

Eicosapentaenoic acid (EPA) ranging from 3% to 10%

Docosahexaenoic acid (DHA) ranging from 3% to 10%

Palmitic acid ranging from 10% to 35%

Oleic acid ranging from 0.5% to 35%

Stearic acid ranging from 0.5% to 9%

- Gamma-linolenic acid ranging from 0.5% to 10%

- Alpha-linolenic acid ranging from 0.5% to 10%

- Azelaic acid ranging from 0.5% to 20%

- Linoleic acid ranging from 0.5% to 35%

Example 3: (for reference only)

- Eicosapentaenoic acid (EPA) ranging from 0.5% to 15% - Docosahexaenoic acid (DHA) ranging from 0.5% to 15% - Palmitic acid ranging from 1% to 35%

- Oleic acid ranging from 0.6% to 35%

- Stearic acid ranging from 0.3% to 15%

- Gamma-linolenic acid ranging from 0.5% to 20%

- Alpha-linolenic acid ranging from 0.5% to 35%

- Azelaic acid ranging from 0.5% to 35%

- Linoleic acid ranging from 0.5% to 35%

Example 4: (for reference only)

- Eicosapentaenoic acid (EPA) ranging from 2% to 12% - Docosahexaenoic acid (DHA) ranging from 2% to 12% - Palmitic acid ranging from 10% to 45%

- Oleic acid ranging between 1% and 35%

- Stearic acid ranging from 0.5% to 25%

- Gamma-linolenic acid ranging from 0.5% to 10%

- Alpha-linolenic acid ranging from 0.5% to 30%

- Azelaic acid ranging from 0.5% to 20%

- Linoleic acid ranging between 3% and 35%

Example 5: (for reference only)

- Eicosapentaenoic acid (EPA) ranging from 35% to 24% - Docosahexaenoic acid (DHA) ranging from 3% to 24% - Palmitic acid ranging from 10% to 40%

- Oleic acid ranging between 15% and 35%

- Stearic acid ranging from 0.5% to 40%

- Gamma-linolenic acid ranging from 0.5% to 25%

- Alpha-linolenic acid ranging from 0.5% to 20%

- Azelaic acid ranging from 0.5% to 25%

- Linoleic acid ranging from 0.5% to 35%

Example 6: (for reference only)

- Eicosapentaenoic acid (EPA) ranging from 1% to 20% - Docosahexaenoic acid (DHA) ranging from 3% to 12% - Palmitic acid ranging from 1% to 30%

- Oleic acid ranging between 2% and 15%

- Stearic acid ranging from 0.5% to 15%

- Gamma-linolenic acid ranging from 0.5% to 15%

- Alpha-linolenic acid ranging from 0.5% to 20%

- Azelaic acid ranging from 0.5% to 10%

- Linoleic acid ranging between 5% and 35%

Example 7 (for reference only)

- Eicosapentaenoic acid (EPA) ranging from 0.5% to 12% - Docosahexaenoic acid (DHA) ranging from 0.5% to 12% - Palmitic acid ranging from 0.5% to 45% %

- Oleic acid ranging between 10% and 35%

- Stearic acid ranging from 0.5% to 10%

- Gamma-linolenic acid ranging from 0.5% to 20%

- Alpha-linolenic acid ranging from 0.5% to 20%

- Azelaic acid ranging from 0.5% to 10%

- Linoleic acid ranging from 0.5% to 15%

Example 8: (for reference only)

- Eicosapentaenoic acid (EPA) ranging from 1% to 12% - Docosahexaenoic acid (DHA) ranging from 1% to 12% - Palmitic acid ranging from 0.5% to 25%

- Oleic acid ranging between 10% and 35%.

- Stearic acid ranging from 0.5% to 10%

- Gamma-linolenic acid ranging from 0.5% to 15%

- Alpha-linolenic acid ranging from 0.5% to 15%

- Azelaic acid ranging from 0.5% to 10%

- Linoleic acid ranging between 2% and 35%

Example 9: (for reference only)

- Palmitic acid 14%

- Linoleic acid 12%

- Oleic acid 15%

_ Stearic acid 14%

- Alpha-linolenic acid 15%

- Gamma-linolenic acid 0.5%

- Azelaic acid 0.5%

- Eicosapentaenoic acid (EPA) 14%

- Docosahexaenoic acid (DHA) 15%

Example 10

- Palmitic acid 20%

- Linoleic acid 10%

- Oleic acid 15%

- Stearic acid 7%

- Alpha-linolenic acid 10%

- N-2-hydroxyethylpalmitamide (PEA) 38%

In vitro evaluation of anti-inflammatory activity

The anti-inflammatory activity of the mixture containing nine fatty acids (see example 9), identified as AS66, has been evaluated by two experimental tests:

1. MTT vitality test to identify the highest non-cytotoxic dose

2. Assessment of the ability of the mixture to reduce the release of pro-inflammatory mediators (IL-6 and IL-8) by means of a human macrophage cell line previously sensitized with LPS

Experimental design

In vivo, the inflammatory reaction is the result of a complex process determined by the response of different cell populations in different regions of the human body. Simplifying, the following phases can be distinguished: external aggression (bacterial or other) accompanied or not by a real tissue injury, release of pro-inflammatory mediators, call of competent cells, production of antibodies, elimination of antigens, activation of the repair and healing process.

Monocytes and macrophages are critical players in both natural and acquired immune responses. Macrophages exist in practically all tissues of the body and show remarkable morphological heterogeneity in different tissues. Monocytes are the blood-borne precursors of macrophages. In the event of infection, tissue damage, or other injury, large numbers of monocytes are recruited from the bloodstream to the site of insult and differentiate in the appropriate macrophage phenotype (see Figure 2).

Macrophages are also important modulators of a variety of immune responses, through the secretion of a wide variety of cytokines and chemokines. Among the mediators produced by monocytes / macrophages, a cytokine (Interleukin 6) and a chemokine (Interleukin 8) were chosen in this study. Interleukin 8 (IL-8) has been chosen for its key role in the primary inflammatory response. It is constitutively produced by different cell populations (monocytes / macrophages, T lymphocytes, endothelial cells, neutrophils, keratinocytes, fibroblasts, and chondrocytes), and this provides it with an important role in "surveillance" and "emergence" operations in tissues.

Interleukin 6 (IL-6) was chosen because it is typical of the late phase of the inflammatory reaction.

IL-6 is a 26 kDa glycoprotein that is not constitutively produced, it is synthesized in response to other pro-inflammatory mediators, specifically "early" such as IL-8, IL-1a and TNF, also called primary cytokines of the inflammatory response.

IL-6 plays a role in both innate (non-specific) and humoral (specific) immune responses. In innate immunity, it stimulates the production of acute phase proteins by liver cells, which are responsible of the appearance of symptoms of systemic allergic reaction, and stimulates the maturation of neutrophils from immature precursors in the bone marrow. In the specific humoral response, it stimulates the proliferation of B lymphocyte clones that produce antibodies specific to the allergen that triggered the reaction.

Cell model

Given the central role of monocytes / macrophages in cell-mediated immune response and their role in modulating the inflammatory response in this study, a human monocyte cell line called THP-1 (human acute monocytic leukemia) was selected. These cells have Fc and C3b receptors, are phagocytic, and grow in suspension that does not adhere to the culture flask.

The cells were purchased from the Istituto Zooprofilattico della Lombardia ed Emilia Romagna (Brescia facility - Italy) and are certified free from bacterial contamination and mycoplasma.

Materials and methods

The inflammatory reaction in THP-1 cells was induced by exposing them to a solution of lipopolysaccharide (LPS), a known inflammatory agent extracted from E. coli, a Gram microorganism, at a concentration of 1ug / ml for two hours.

o Culture medium: RPMI 1640, supplemented with 2 mM glutamine, 10% supplemented fetal calf serum, non-essential amino acids, 2-mercaptoethanol (0.05 mM) and a mixture of penicillin antibiotics 2000 IU / ml, streptomycin 1000 IU / ml, Fungizone 2ug / ml (Euroclone).

o MTT stock solution: 5 mg / ml in water. (Sigma Aldrich)

o MTT working solution: 1 mg / ml in RPMI 1640

o Overlay buffer: phosphate buffer saline (PBS)

o Assay buffer: PBS with 4% bovine serum albumin (BSA)

o Wash buffer: 50 mM Tris, 0.2% Tween 20

o TMB substrate dissolution

o Stop solution: H ² SO ⁴ 0.18 M

1. Identification of the highest non-cytotoxic dose

In general, the first choice of non-cytotoxic dose is the dose at which the residual% cell viability does not differ by more than 15% from the negative control viability.

Day 1: Seeding cells

After verifying the vitality of the cells in culture, they were collected and centrifuged (10 minutes at 900 rpm), the pellet was resuspended in a suitable volume of RPMi 1640 and the cells were counted with an electronic cell counter (Scepter - Millipore). Subsequently, a cell suspension was prepared at a density of 400,000 cells / ml. This suspension was distributed into 24 microwell plates at 2: 1 ml of suspension per well (400,000 cells / well). The plates were then placed in an incubator at 37 ° C and the atmosphere was enriched with 5% CO ² until the next day (20-22 hours).

Day 2:

Sample preparation:

1 g of sample (mixture of Example 9) was weighed and resuspended in a sufficient volume of ethanol to obtain a final concentration of 5 mg / ml. From this stock solution a series of dilutions (2x) from 2 mg / ml to 0.312 mg / ml were prepared in RPMI medium with 5% FBS.

Inoculation of the sample: after checking the health status and density of the cells seeded on day 1, a volume of 0.5 ml of culture medium was collected from each well, which was later replaced with the same volume of dilutions 2x pre-prepared samples. 6 different doses of the sample were inoculated: from 1 mg / ml and 5 additional double dilutions of the stock solution were placed in medium with 5% FBS. The plates were then placed in an incubator at 37 ° C and the atmosphere was enriched with 5% CO ² for 20-22 hours. The negative control consists of cells exposed only to culture medium that represents 100% of vitality. Day 3:

After verifying the health status and density of the cells exposed to the sample, the MTT vitality test was performed.

MTT vitality test

MTT is a tetrazolium salt that, in its oxidized form, is soluble and yellow in color. It is converted to its reduced form of formazan salt by cytoplasmic and mitochondrial dehydrogenases (succinate dehydrogenases), in this form the salt is insoluble and precipitates to form purplish blue crystals. The addition of isopropanol solubilizes the crystals, forming a violet solution whose strength, evaluated with a spectrophotometer reading of 540 ± 30 nm, is proportional to the amount of living cells present at the time the test is performed.

The inoculum was removed and replaced with the same volume of the MTT working solution.

Plates were placed in an incubator for three hours. At the end of the incubation period, the salt has been removed and replaced with the same volume of isopropanol. Then, after 5 minutes of stirring, the optical density was read at 570 nm (± 30 nm) with a spectrophotometer (Victor X5-Perkin-Elmer).

From the analysis of the results obtained, sample concentrations of 0.1, 0.06 and 0.03 mg / ml were selected for the anti-inflammatory test.

2. Evaluation of anti-inflammatory activity

Day 1: Seeding cells

After verifying the vitality of the cells in culture, they were collected and centrifuged (10 minutes at 900 rpm), then the pellet was resuspended in a suitable volume of RPMI 1640 and the cells were counted with an electronic cell counter (Scepter - Millipore). Subsequently, a cell suspension was prepared at a density of 400,000 cells / ml. This suspension was distributed in Petri dishes: 3 ml of cell suspension / ml (1,200,000 cells / plate). The plates were then placed in an incubator at 37 ° C and the atmosphere was enriched with 5% CO ² until the next day (20-22 hours).

Day 2:

Sample preparation:

1 g of powder (mixture of Example 9) was weighed out and resuspended in a sufficient volume of ethanol to obtain a final concentration of 5 mg / ml. From this stock solution, three dilutions (2x) were prepared: 0.2, 0.12 and 0.06 mg / ml in RPMI medium with 5% FBS.

Quality control (QC):

CC was represented by a solution of 0.1% Locoidon at 1 mg / ml, a known anti-inflammatory product containing cortisol. The solution was prepared in RPMI 1640 medium with 5% FBS.

Positive control

The positive control was represented by cells exposed to the 1 pg / ml LPS solution.

Induction of the inflammatory reaction:

After one night of culture, the plates were removed and approximately half of the culture medium was replaced with the same volume of the 2 pg / ml LPS solution (final concentration 1 pg / ml). LPS exposure was continued for two hours.

Sample inoculation:

At the end of the incubation time with LPS, after checking the health status of the cells, the 1 Ps solution was replaced with the same volume of the different doses of the previously prepared sample. The plates were then placed in an incubator at 37 ° C and the atmosphere was enriched with 5% CO ² for 20-22 hours. Day 3

After checking the health status of the treated cells, the cell suspension was collected in 1.5 ml tubes and centrifuged (1000 g for 10 minutes). Then, the ELISA test was carried out in order to evaluate the concentrations of IL-6 and IL-8 in the culture medium.

Finally, in order to exclude false positives, that is, to avoid classifying the lack of living and vital cells as an anti-inflammatory effect, after centrifugation, the cells were subjected to the MTT cell viability test. Results

Cell survival percentage

% vitality - [(OD sample / OD control neg.) X 100]

% Vitality Inhibition - (100 -% Vitality)

O.D .: optical density

Sample OD: optical density obtained from cells exposed to the sample

Negative control OD: optical density obtained from cells exposed only to the culture medium (100% vitality) Assessment of the levels of IL-6 and IL-8 secreted in the culture medium

Anti-inflammatory efficacy

% inhibition of IL-8 - (100 - (pg / ml IL-8 sample / pg / ml IL-8cntrl pos) * 100) pg / ml IL-8 sample: pg / ml IL-8 secreted by cells exposed to the LPS solution and then to various sample dilutions

pg / ml I L-8cntrl pos: pg / ml of IL-8 secreted by cells exposed only to the LPS solution.

% inhibition of IL-6 - (100 - (pg / ml IL-6 sample / pg / ml I L-6cntrl pos) * 100) pg / ml IL-6 sample: pg / ml IL-6 secreted by cells exposed to the LPS solution and then to various sample dilutions

pg / ml of IL-6cntrl pos: pg / ml of IL-8 secreted by cells exposed only to the LPS solution.

Test acceptance criteria

The test meets the acceptance criteria of the method if the equation of the regression line of the standard curve has a coefficient of determination (R2)> 0.8 (between 0.8 and 1).

The coefficient of variation of the raw data must be <20%

P-value (T test): <0.05

a) IC ⁵⁰

IC ⁵⁰ - 0.8 mg / ml (see figure 3)

Table 1

IC ⁵⁰ corresponds to the dose of the sample that kills 50% of the cells in the incubation period. b) IL-8

Table 2

The standard curve of IL-8 and the percentage of inhibition of IL-8 are represented in Figure 4.

Table 3

c) IL-6

Table 4

The standard curve of IL-6 and the percentage of inhibition of IL-6 are represented in figure 5.

Table 5

As can be seen, none of the doses tested showed cytotoxic action that could distort the result of the evaluation of the anti-inflammatory activity of the AS66 sample tested (mixture of example 9).

All the doses of AS66 analyzed have been shown to be capable of inhibiting the release of both cytokines analyzed.

The tests carried out have identified a maximum effective dose equivalent to 0.03 mg / ml: at this concentration, the anti-inflammatory activity of AS66 determines an inhibition of IL-6 and IL-8 release equal to 51% and 66.7% respectively.

In other words, lower concentrations of the mixture according to the present invention lead to a higher anti-inflammatory activity, in the absence of cytotoxic effects.

The same two experimental tests were also carried out on a composition comprising a mixture of five fatty acids and N-2-hydroxyethylpalmitamide (PEA) (see example 10), identified as LC88.

As in the previous test, the IC ⁵⁰ has been identified and is 0.38 mg / ml (see Figure 6).

The data obtained with respect to the percentage of IL-6 inhibition are presented in the table below and in Figure 7.

Table 6

As can be easily appreciated from the above data, none of the doses showed cytotoxic action that could distort the result of the evaluation of the anti-inflammatory activity of the sample under test.

All doses of LC88 have been shown to be capable of inhibiting IL-6 release with a significant effect dose / response.

In vivo testing

1. In vivo tests on animals

Several studies have been carried out in animals in order to confirm the efficacy and safety of the mixture of the present invention (see example 9), in particular for the control of the symptoms in the external ears of pets (cats, dogs).

From April 2013 to October 2013, 33 specimens of both cats and dogs belonging to different breeds were registered, with many different pathologies in the external auditory canal. In particular, 23 dogs and 10 cats have been observed.

Regarding dogs: 5 mastiffs, 8 hunting dogs, 10 terriers.

In relation to cats, 10 cats have been observed, of which 8 are European and 2 are Siamese, living in two colonies.

All dogs have been divided on the basis of etiopathogenesis / pathology and breed membership and FCI (Federation Cynological International) classification.

Study 1: clinical study in dogs belonging to class 2: Mastiff of many breeds suffering from Malassezia ( Pityrosporum)

5 Mastiff dogs, aged between 8 months and 5 years, three males and two females

At the first visit, the external auditory canal was edematous and erythematous in both ears, with the presence of waxy material and a bad odor. Ear cytology reveals the presence of Malassezia in a very large number for each sample. The therapy provides the use of a chlorhexidine-based veterinary ear disinfectant to be administered twice daily, morning and evening, for 15 days. At the second visit, ie 10 days later, the administration of fatty acid-based ear drops was added because the growing yeast had been controlled, but the ear canal was still erythematous with persistent symptoms in the animal. After administration of ear drops based on the mixture of example 9, the next control after 4 days showed that the external auditory canal normalized without erythema and without any symptoms shown by the animals.

Conclusions: In normal practice, at least 40% of the dogs should have had a yeast exacerbation ( Pityrosporum Ovale or Malassezia), in the other 40% the normalization of the tissue would have taken at least 15/30 days, in the The remaining 20% healing and normalization of the tissue would have occurred in a period of 30/45 days. The ear drops based on the mixture of Example 9 allowed total remission of inflammation and healing in a time period of only 3/4 days in all treated specimens. It is very important to emphasize the control of inflammation in the tissues, since this approach avoids the stimulus of scratching thus avoiding the exacerbation of the pathology and allowing a very rapid healing.

Study 2: Clinical cases in dogs belonging to class 7 (hunting dogs): foreign body etiopathogenesis:

8 dogs were considered, 5 Setter, 3 Pointer, with ages between 2 and 5 years, 5 males and 3 females, taken to the veterinary clinic for an ear problem.

The presence of a foreign body was highlighted in the external auditory canal. After having removed said foreign body and having verified the integrity of the tympanic membrane, after cleaning the auditory meatus, an ear product based on the mixture of example 9 was administered for 5 days twice a day in the morning and at night in the affected ear. After 5 days of treatment, the control visit showed complete normalization of the tissues and the disappearance of the erythema.

Conclusions: In usual practice, 30% of the animals would have developed a superinfection and would have been treated with antibiotics, 40% would have had persistent inflammation for at least 20/30 days after removal of the foreign body and a healing in a time of between 30 and 40 days, in the other 30% of the animals an almost normalization in 20 days. With the use of fatty acid-based ear drops in the postoperative phase (removal of foreign bodies), the inflammation has been controlled, allowing the complete normalization of the tissues in 4/5 days, avoiding the appearance of a superinfection.

Study 3: clinical cases in dogs belonging to class 3, Terrier, etiopathogenesis of atopic dermatitis, allergies and hormonal alterations.

10 dogs have been observed, 5 West Highlands White Terriers, 3 Jack Russel Terriers, 2 Yorkshire Terriers, aged between 1 and 10 years, 7 females and 3 males.

All animals were affected by allergic dermatitis, atopic dermatitis with symptomatic otitis. Some dogs (5) at the visit showed a presence of exudate with skin scales in the external atrium. The mixture of Example 9 has been administered as a single therapeutic device. The results were amazing; In just 3 days the situation improved significantly until having almost normal skin, which allowed the possibility of an in-depth examination with an otoscope.

Conclusions: Otitis due to allergic dermatitis is quite common in 15% of the canine population. The single use of the mixture of the present invention allowed the resolution of the pathology, with the normalization of the tissues in a very short time, only 3/4 days.

Study 4: Clinical cases in European and Siamese cats: etiopathogenesis of ear infections by parasites

10 cats, 8 Europeans and 2 Siamese, 7 females and 3 neutered males, aged between 6 months and 5 years, brought to visit for an otitis that showed the parasitic nature. All cats showed abundant material in both ear canals with the presence of ticks and severe itching, and 3 of them developed otohematoma. To the spot-on salamectin therapy, a fatty acid-based product for tissue normalization had been added which determined optimal resolution in just 2/3 days.

Conclusions: in 70% of the animals in the same pathological conditions, the edema and itching would have persisted for many days up to 20/30 days, thus determining the additional damage to the already inflamed tissues. The administration of the mixture according to example 9 allowed a normalization of the tissue in a very short time, normalizing both the erythematous and inflammatory process, and eliminating the itching sensation.

Conclusion of studies:

A very rapid resolution of the pathologies was observed until the disappearance of the symptoms in a very short time. Therefore, the mixture of the present invention should be considered a valid therapeutic instrument for the control and advancement of inflammatory processes of various etiologies of pathologies that affect the external ear of pets.

These results have been confirmed by an additional clinical study of a periophthalmic cream based on the mixture of example 9 for the treatment of pets with periophthalmic pathologies, all the specimens that participated in the study and the related pathologies are presented in the table below.

Table 7

All cats and dogs received a treatment with an ophthalmic cream based on the mixture of the present invention, the state of symptoms resolved in an average time of 5 days (from 3 to 9 days) with an average application of twice a day .

The periophthalmic cream based on 9 fatty acids, proved to be effective in the control and advance of the symptoms related to inflammation of a varied nature in the eyelids of dogs and cats.

2. In vivo tests in humans

The following data refers to a comparative study of GutLife and EvaLife vaginal cream, both containing the mixture of the present invention and PEA, for administration to patients receiving chemoradiation for anus / anal canal SCC. This study was conducted at the Southampton Cancer Center.

Until recently, there have been a limited number of products available for this group of patients to use for any skin reactions that develop while undergoing radiation therapy.

GutLife and EvaLife (pH balanced for women) are creams that contain the mixture of the present invention (see example 10) and that can be used for the treatment of changes in the skin / mucous membranes (itching, burning, redness, swelling and sensitivity) in the anal and perianal area in patients undergoing radiation or chemotherapy.

GutLife is an anal and perianal cream that can be used by both male and female patients. EvaLife is specifically for use by female patients around the vagina and vulva, it is pH balanced to be most beneficial for female patients.

study

It is proposed that GutLife and EvaLife be tested to assess their benefit to the patient and whether it reduces hospital admissions for this group of patients. The study uses the r To G scale for skin assessment as advised by the Society of Radiographers and used at the Southampton Oncology Center. The physician and / or colorectal radiographer assessed and recorded it weekly. Pain scores and RTOG were used for four patients (two men and two women) as a control group compared to a 2nd group using GutLife and EvaLife products. Assessments were performed at the same weekly review clinic each week during the patient's course of treatment. Results can be summarized and compiled in a table

Dosage prescription

Phase I: 30.6 Gy at MPD (ICRU reference point) in 17 fractions

Phase II: 19.8 Gy at 100% for compliant plan (ICRU reference point) in 11 daily fractions. Maximum dose 107%, minimum 95% within PTV (Planning Target Volume).

Or: 19.8 Gy to MPD in 11 daily fractions if parallel opposing fields

Concurrent chemotherapy

Patients have received concurrent chemotherapy through a PICC (peripherally inserted central catheter) line as an outpatient.

Mitomycin C 12mg / m2 Bolus IV Day 1 (maximum 20 mg) 5-Fluorouracil 1000mg / m2 / day continuous infusion days 1-4 (week 1) and day 29-32 (week 5).

Chemotherapy was given before receiving the first fraction of radiation therapy. The RTOG skin assessment tool was used as advised by the Society of Radiographers to assess skin reaction (see Table 8)

Table 8

Patients underwent radical radiotherapy treatment for anal / anal canal SCC with concurrent chemotherapy from April 2012 - April 2013 at the Southampton Cancer Center. In this period, 28 female patients and 11 male patients were treated.

From the data collected during the last 12 months, there were 39 patients treated during this time, of which there were 6 confirmed admissions to the center for cutaneous adverse reactions attributed to radiotherapy, of this group 4 were female patients and 2 were male.

Table 9: Results in patients with anal cancer (control group)

Table 10: Results in patients with anal cancer (test group)

Summary

Due to the limited availability of GutLife and EvaLife, the products could only be used for four patients. Although limited in number of patients, there are still valid observations and indications from this study.

The maximum RTOG score for all patients in the control group was 3 compared to two patients who scored 3 (1 male, 1 female) using GutLife and EvaLife also the highest RTOG score for patients using GutLife / EvaLife was occurs later in treatment compared to the control group. This would suggest that GutLife and EvaLife act preventively.

There appears to be a clear contrast between the maximum pain scores of the control group and the GutLife / EvaLife group. The highest pain score recorded for the control group was 8 (recorded in two patients) with a lower score of 6 (recorded in 1 patient) compared to a high score of 7 in the study group (recorded in 1 patient), the lowest pain score recorded in the test group was 4.

One patient was admitted to hospital from the control group due to skin reaction developed from treatment. One patient was admitted to the study group hospital, but this was not due to a skin reaction.

From this limited study, patients using GutLife and EvaLife cream managed radiation therapy-induced skin reaction better than those in the control group, reporting lower pain score on average, lower maximum RTOG score , as well as delaying the appearance of the skin reaction until the last two weeks of treatment. Based on these favorable results, anal cancer radiation therapy specialists would support a larger multicenter trial and / or include GutLife and EvaLife cream to be used for routine skin care of anal cancer patients undergoing radiation therapy at the Center for Southampton Oncology.

Claims (19)

1. Pharmaceutical, cosmetic and / or dietary composition comprising a mixture of at least five fatty acids selected from palmitic acid, oleic acid, stearic acid, linoleic acid, alpha-linolenic acid, gamma-linolenic acid, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), azelaic acid and myristic acid and the physiologically stable excipient N-2-hydroxyethylpalmitamide. 2. Pharmaceutical, cosmetic and / or dietetic composition according to any of claim 1, comprising a mixture characterized in that it comprises five fatty acids, such as palmitic acid, oleic acid, stearic acid, linoleic acid and alpha-linolenic acid. 3. Pharmaceutical, cosmetic and / or dietetic composition according to any of claim 1, comprising a mixture characterized in that it comprises eight fatty acids, such as palmitic acid, oleic acid, stearic acid, linoleic acid, alpha linolenic acid, gamma linolenic acid , eicosapentaenoic acid and docosahexaenoic acid. 4. Pharmaceutical, cosmetic and / or dietetic composition according to any of claim 1, comprising a mixture characterized in that it comprises nine fatty acids, such as palmitic acid, oleic acid, stearic acid, linoleic acid, alpha-linolenic acid, gamma-linolenic acid. , eicosapentaenoic acid, docosahexaenoic acid and azelaic acid. Pharmaceutical, cosmetic and / or dietetic composition according to any of claim 1, characterized in that palmitic acid is contained in said mixture in an amount by weight comprised between 0.2% and 48%; oleic acid is contained in said mixture in an amount by weight comprised between 0.2% and 38%; stearic acid is contained in said mixture in an amount by weight comprised between 0.2% and 42%; linoleic acid is contained in said mixture in an amount by weight comprised between 0.2% and 40%; alpha linolenic acid is contained in said mixture in an amount by weight comprised between 0.2% and 38%; gamma linolenic acid is contained in said mixture in an amount by weight comprised between 0.2% and 30%; eicosapentaenoic acid is contained in said mixture in an amount by weight comprised between 0.2% and 25%; docosahexaenoic acid is contained in said mixture in an amount by weight comprised between 0.2% and 25%; azelaic acid is contained in said mixture in an amount by weight comprised between 0.2% and 40%; myristic acid is contained in said mixture in an amount by weight comprised between 0.005% and 0.01%, relative to the total weight of the mixture. Composition according to claim 1, characterized in that palmitic acid is contained in said composition in an amount by weight comprised between 5% and 45%; oleic acid is contained in said composition in an amount by weight comprised between 2% and 35%; stearic acid is contained in said composition in an amount by weight of between 2% and 35%; linoleic acid is contained in said composition in an amount by weight comprised between 1% and 40%; alpha linolenic acid is contained in said composition in an amount by weight comprised between 1% and 25%; gamma linolenic is contained in said composition in an amount by weight comprised between 0.5% and 10%; eicosapentaenoic acid is contained in said composition in an amount by weight comprised between 1% and 20%; docosahexaenoic acid is contained in said composition in an amount by weight comprised between 1% and 25%, azelaic acid is contained in said composition in an amount by weight comprised between 0.5% and 10%, the acid Myristic is contained in said composition in an amount by weight comprised between 0.05% and 1%, with respect to the total weight of the composition. Composition according to any one of claims 1-7, characterized in that it is in oral, topical, rectal, vaginal, ophthalmic or parenteral form. Composition according to claim 7, characterized in that said oral form is selected from tablet, capsule, granule, oil bead, solution, suspension, carrier and aerosol, preferably said oral form is tablet, capsule or solution. Composition according to claim 7, characterized in that said topical form is selected from cream, ointment, gel, solution, suspension, spray, patch and granular lyophilized powder, preferably said topical form is cream, gel, spray, ointment or lyophilized powder granular. Composition according to claim 7, characterized in that said parenteral form is selected from aqueous buffer solution and oily suspension, preferably said parenteral form is oily suspension. Composition according to claim 7, characterized in that said rectal form is a suppository, an enema or a solution for endocavitary use. Composition according to claim 7, characterized in that said vaginal form is an ovule, a catheter, a gel or a solution for endocavitary use. Composition according to claim 7, characterized in that said ophthalmic form is an eye drop, an eye wash, a cream or an ointment. Composition according to any one of claims 1-13, characterized in that the mixture according to any one of claims 1-5 is present in said composition in an amount by weight comprised between 10% and 60%, preferably between 15% and 45% with respect to the total weight of the composition. Composition according to claim 1, characterized in that it is a dermocosmetic composition, a medicine, a dietary supplement or a medical device. 16. Pharmaceutical composition according to any one of claims 1-15, for use in the treatment of inflammatory pathologies, preferably said inflammatory pathologies are in the acute or chronic phase. 17. Pharmaceutical composition for use according to claim 16, characterized in that said inflammatory pathologies are selected dermatological pathologies of atopic dermatitis, dermatomyositis, scleroderma, psoriasis, polymyositis, pemphigoid and epidermolysis bullosa pemphigoid, or ophthalmic pathologies selected from Sjogren's syndrome, ophthalmia sympathetic, uveitis and uveo-retinitis, or selected mucosal diseases from inflammation of the gastrointestinal mucous membranes (Crohn's disease) and inflammation of the oral and genital mucosa, or joint and connective pathologies selected from rheumatoid arthritis, psoriatic arthritis, lupus erythematosus and discoid and systemic lupus erythematosus, or chronic pathological inflammation selected from chronic solar dermatitis, asthma and intestinal and pulmonary fibrosis, and chronic arthritis, or degenerative diseases of the peripheral nervous system and of the central nervous system selected from multiple sclerosis, disorders n autoimmune eurodegenerative, non-autoimmune neurodegenerative disorders, inflammatory processes associated with the central nervous system such as Parkinson's disease, senile dementia, bacterial meningitis, HIV infection and traumatic injury, and peripheral nervous system diseases such as inflammatory radiculopathies, or diseases of the system central and peripheral nervous where inflammatory processes follow the first insult of ischemic origin, such as neuropathy due to compression and traumatic neuropathy, stroke and traumatic brain injury, or cardiological diseases derived from perfusion phenomena as a result of ischemic damage. 18. Pharmaceutical composition for use according to claim 17, characterized in that it is administered to a human being or an animal, preferably when administered to an animal, said mixture and / or composition is used for the treatment of neurogenic inflammation, joint and connective diseases such as laminitis in horses, arthritis, respiratory pathologies, ophthalmic inflammation, keratoconjunctivitis sicca and allergic inflammatory manifestation. 19. Pharmaceutical composition for use according to claim 18, characterized in that it is administered daily, from one to four doses a day, preferably to be administered for at least 4 weeks, more preferably said dose contains from 0.1 to 50 mg of composition / kg of patient body weight, preferably from 0.5 to 20 mg / kg of patient body weight.